Plant Molecular Biology

, Volume 46, Issue 6, pp 705–715 | Cite as

Tissue-specific and developmental-specific expression of an Arabidopsis thaliana gene encoding the lipoamide dehydrogenase component of the plastid pyruvate dehydrogenase complex

  • Sinead C. Drea
  • Ruth M. Mould
  • Julian M. Hibberd
  • John C. Gray
  • Tony A. Kavanagh


We describe an Arabidopsis thaliana gene, ptlpd2, which codes for a protein with high amino acid similarity to lipoamide dehydrogenases (LPDs) from diverse species. Ptlpd2 codes for a precursor protein possessing an N-terminal extension predicted to be a plastid-targeting signal. Expression of the ptlpd2 cDNA in Escherichia coli showed the encoded protein possessed the predicted LPD activity. PTLPD2 protein, synthesized in vitro, was efficiently imported into isolated chloroplasts of Pisum sativum and shown to be located in the stroma. In addition, fusion proteins containing the predicted transit peptide of PTLPD2 or the entire protein fused at the N-terminus with the green fluorescent protein (GFP), showed accumulation in vivo in chloroplasts but not in mitochondria of A. thaliana. Expression of ptlpd2 was investigated by introducing ptlpd2 promoter-β-glucuronidase (GUS) gene fusions into Nicotiana tabacum. GUS expression was observed in seeds, flowers, root tips and young leaves. GUS activity was highest in mature seeds, decreased on germination and increased again in young leaves. Expression was also found to be temporally regulated in pollen grains where it was highest in mature grains at dehiscence. Database searches on ptlpd2 sequences identified a second A. thaliana gene encoding a putative plastidial LPD and two genes encoding proteins with high similarity to the mitochondrial LPD of P. sativum.

Arabidopsis thaliana chloroplast import GFP fusion protein lipoamide dehydrogenase transit peptide. 


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  1. Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D.J. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucl. Acids Res. 25: 3389–3402.CrossRefPubMedGoogle Scholar
  2. Bao, X., Focke, M., Pollard, M. and Ohlrogge, J. 2000. Understanding in vivo carbon precursor supply for fatty acid biosynthesis in leaf tissue. Plant J. 22: 39–50.PubMedGoogle Scholar
  3. Benen, J.A.E., van Berkel, W.J.H., Veeger, C. and de Kok, A. 1992. Lipoamide dehydrogenase from Azotobacter vinelandii:the role of the C-terminus in catalysis and dimer stabilization. Eur. J. Biochem. 207: 495–505.Google Scholar
  4. Bevan, M. 1984. Binary Agrobacterium vectors for plant transformation. Nucl. Acids Res. 12: 8711–8721.PubMedGoogle Scholar
  5. Bevan, M., Bancroft, I., Bent, E. et al. 1998. Analysis of 1.9 Mb of contiguous sequence from chromosome 4 of Arabidopsis thaliana. Nature 391: 485–488.PubMedGoogle Scholar
  6. Bourguignon, J., Macheral, D., Neuberger, M. and Douce, R. 1992. Isolation, characterization and sequence analysis of a cDNA clone encoding L-protein, the dihydrolipoamide dehydrogenase component of the glycine cleavage system from pea-leaf mitochondria. Eur. J. Biochem. 204: 865–873.PubMedGoogle Scholar
  7. Bourguignon, J., Merand, V., Rawsthorne, S., Forest, E. and Douce, R. 1996. Glycine decarboxylase and pyruvate dehydrogenase complexes share the same dihydrolipoamide dehydrogenase in pea leaf mitochondria: evidence from mass spectrometry and primary-structure analysis. Biochem. J. 313: 229–234.PubMedGoogle Scholar
  8. Bowman, S.B., Zaman, Z. Collinson, L.P., Brown, A.J. and Dawes, I.W. 1992. Positive regulation of the LPD1 gene of Saccharomyces cerevisiae by the HAP2/HAP3/HAP4 activation system. Mol. Gen. Genet. 231: 296–303.Google Scholar
  9. Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248–255.CrossRefPubMedGoogle Scholar
  10. Busk, P.K. and Pagès, M. 1998. Regulation of abscisic acid-induced transcription. Plant Mol. Biol. 37: 425–435.PubMedGoogle Scholar
  11. Camp, P.J. and Randall, D.D. 1985. Purification and characterization of the pea chloroplast pyruvate dehydrogenase complex. Plant Physiol. 77: 571–577.Google Scholar
  12. Conner, M., Krell, T. and Lindsay, J.G. 1996. Identification and purification of a distinct dihydrolipoamide dehydrogenase from pea chloroplasts. Planta 200: 195–202.PubMedGoogle Scholar
  13. Dastoor, F.P., Forrest, M.E. and Beatty, J.T. 1997. Cloning, se-quencing and oxygen regulation of the Rhodobacter capsulatus alpha-ketoglutarate dehydrogenase operon. J. Bact. 179: 4559–4566.PubMedGoogle Scholar
  14. Davis, S.J. and Vierstra, R.D. 1998. Soluble, highly fluorescent variants of green fluorescent protein (GFP) for use in higher plants. Plant Mol. Biol. 36: 521–528.CrossRefPubMedGoogle Scholar
  15. Denyer, K. and Smith, A.M. 1988. The capacity of plastids from developing pea cotyledons to synthesize acetyl-CoA. Planta 173: 172–182.Google Scholar
  16. Emanuelsson, O., Nielsen, H. and von Heijn, G. 1999. ChloroP, a neural network-based method for predicting chloroplast transit peptides and their cleavage sites. Protein Sci. 8: 978–984.PubMedGoogle Scholar
  17. Engels, A. and Pistorius, E.K. 1997. Characterization of a gene encoding dihydrolipoamide dehydrogenase of the cyanobacterium Synechocystis sp. strain PCC 6803. Microbiology 143: 3543–3553.PubMedGoogle Scholar
  18. Faure, M., Bourguignon, J., Neuburger, M., Macherel, D., Sieker, L., Ober, R., Kahn, R., Cohen-Addad, C. and Douce, R. 2000. Interaction between the lipoamide-containing H-protein and the lipoamide dehydrogenase (L-protein) of the glycine de-carboxylase multienzyme system. 2. Crystal structures of H-and L-proteins. Eur. J. Biochem. 267: 2890–2898.PubMedGoogle Scholar
  19. Foster, R., Izawa, T. and Chua N.H. 1994. Plant b-ZIP proteins gather at AGCT elements. FASEB J. 8: 192–200.PubMedGoogle Scholar
  20. Gavel, Y. and von Heijne, G. 1990. A conserved cleavage-site motif in chloroplast transit peptides. FEBS Lett. 261: 455–458.PubMedGoogle Scholar
  21. Gray, M.W. 1999. Evolution of organellar genomes. Curr. Opin. Genet. Dev.9: 678–687.PubMedGoogle Scholar
  22. Harwood, J.L. 1996. Recent advances in biosynthesis of fatty acids. Biochim. Biophys. Acta 1301: 7–56.PubMedGoogle Scholar
  23. Hibberd, J.M, Linley, P.J., Khan, M.S. and Gray, J.C. 1998. Transient expression of green fluorescent protein in various plastid types following microprojectile bombardment. Plant J. 16: 627–632.Google Scholar
  24. Higo, K., Ugawa, T., Iwamoto, M. and Higo, H. 1998. PLACE: a database of plant cis-acting regulatory DNA elements. Nucl. Acids Res. 26: 358–359.PubMedGoogle Scholar
  25. Horsch, R.B., Fry, J.E., Hoffman, N.L., Eicholtz, D., Rogers, S.G. and Fraley, R.T. 1985. A simple and general method for transferring genes into plants. Science227: 1229–1231.Google Scholar
  26. Jefferson, R.A., Kavanagh, T.A. and Bevan, M.W. 1987. GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6: 3901–3907.PubMedGoogle Scholar
  27. Johnston, M.L., Luethy, M.H., Miernyk, J.A. and Randall, D.D. 1997. Cloning and molecular analyses of the Arabidopsis thaliana plastid pyruvate dehydrogenase subunits. Biochim. Biophys. Acta 1321: 200–206.PubMedGoogle Scholar
  28. Johnston, M.L., Miernyk J.A. and Randall, D.D. 2000. Import, processing, and assembly of the α-and β-subunits of chloroplast pyruvate dehydrogenase. Planta 211: 72–76.PubMedGoogle Scholar
  29. Kang, F. and Rawsthorne, S. 1994. Starch and fatty acid synthesis in plastids from developing embryos of oilseed rape (Brassica napus L.). Plant J. 6: 795–805.Google Scholar
  30. Ke, J., Behal, R.H., Back, S.L., Nikolau, B.J., Wurtele, E.S. and Oliver, D.J. 2000. The role of pyruvate dehydrogenase and acetyl-coenzyme A synthase in fatty acid synthesis in developing Arabidopsis seeds. Plant Physiol. 123: 497–508.PubMedGoogle Scholar
  31. Kimura, M. 1980. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequence. J. Mol. Evol. 16: 111–120.PubMedGoogle Scholar
  32. Lutziger, I. and Oliver, D.J. 2000. Molecular evidence of a unique lipoamide dehydrogenase in plastids: analysis of plastidic lipoamide dehydrogenase from Arabidopsis thaliana. FEBS Lett. 484: 12–16.PubMedGoogle Scholar
  33. Mattevi, A., Schierbeek, A.J. and Hol, W.G. 1991. The refined crystal structure of Azotobacter vinelandii lipoamide dehydrogenase at 2.2 Å resolution. A comparison with the structure of glutathione reductase. J. Mol. Biol. 220: 974–995.Google Scholar
  34. Mattevi, A., Obmolova, G., Kalk, K.H., van Berkel, W.J. and Hol, W.G. 1993. Three-dimensional structure of lipoamide dehydrogenase from Pseudomonas fluorescens at 2.8 Å resolution. Analysis of redox and thermostability properties. J. Mol. Biol. 230: 1200–1215.PubMedGoogle Scholar
  35. Mooney, B.P., Miernyk, J.A. and Randall, D.D. 1999. Cloning and characterization of the dihydrolipoamide S-acetyltransferase subunit of the plastid pyruvate dehydrogenase complex (E2) from Arabidopsis. Plant Physiol. 120: 443–452.PubMedGoogle Scholar
  36. Mould, R.M. and Gray, J.C.1998a. Preparation of chloroplasts for protein synthesis and protein import. In: J.E. Celis (Ed.) Cell Biology: A Laboratory Handbook, vol. 2, Academic Press, New York, pp. 81–86.Google Scholar
  37. Mould, R.M. and Gray, J.C. 1998b. Import of nuclear-encoded proteins by isolated chloroplasts and thylakoids. In: J.E. Celis (Ed.) Cell Biology: A Laboratory Handbook, vol. 2, Academic Press, New York, pp. 286–292.Google Scholar
  38. Nakai, K. and Kanehisa, M. 1992. A knowledge base for predicting protein localization sites in eukaryotic cells. Genomics. 14: 897–911.Google Scholar
  39. Patel, M.S. and Roche, T.E. 1990. Molecular biology and biochemistry of pyruvate dehydrogenase complexes. FASEB J. 4: 3224–3233.PubMedGoogle Scholar
  40. Stevens R.G., Creissen, G.P. and Mullineaux, P.M. 1997. Cloning and characterization of a cytosolic glutathione reductase cDNA from pea (Pisum sativum L.) and its expression in response to stress. Plant Mol. Biol. 35: 641–654.Google Scholar
  41. Tatusova, T.A. and Madden, T.L. 1999. BLAST 2 sequences: a new tool for comparing protein and nucleotide sequences. FEMS Microbiol. Lett. 174: 247–250.CrossRefPubMedGoogle Scholar
  42. Taylor, A.E., Cogdell, R.J. and Lindsay, J.G. 1992. Immunological comparison of the pyruvate dehydrogenase complexes from pea mitochondria and chloroplasts. Planta 188: 225–231.Google Scholar
  43. Toyoda, T., Suzuki, K., Sekiguchi, T., Reed, L.J. and Takaneka, A. 1998. Crystal structure of eukaryotic E3, lipoamide dehydrogenase from yeast. J. Biochem. 123: 668–674.PubMedGoogle Scholar
  44. Twell, D., Yamaguchi, J., Wing, R.A., Ushiba, J. and McCormick, S. 1991. Promoter analysis of genes that are coordinately expressed during pollen development reveals pollen-specific enhancer sequences and shared regulatory elements. Genes Dev. 5: 496–507.PubMedGoogle Scholar
  45. Zaman, Z., Bowman, S.B., Kornfeld, G.D., Brown, A.J. and Dawes, I.W. 1999. Transcription factor GCN4 for control of amino acid biosynthesis also regulates the expression of the gene for lipoamide dehydrogenase. Biochem. J. 340: 855–862.PubMedGoogle Scholar

Copyright information

© Kluwer Academic Publishers 2001

Authors and Affiliations

  • Sinead C. Drea
    • 1
  • Ruth M. Mould
    • 2
  • Julian M. Hibberd
    • 2
  • John C. Gray
    • 2
  • Tony A. Kavanagh
    • 1
  1. 1.Department of GeneticsTrinity CollegeDublin 2Ireland
  2. 2.Department of Plant SciencesUniversity of CambridgeCambridgeUK

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